The goal of these studies is to investigate molecular mechanisms regulating the activation of factor IX via the extrinsic pathway of coagulation and to further delineate structure-function relationships in factors IX and VII. By a select number of site directed mutagenesis as well as homologous domain replacement studies, we will further delineate the functions of the EGF-1, EGF-2 and the protease domains of factor IX/IXa and factor VIIVIIa. The homologous domains are hypothesized to be substantially different from the corresponding domains in the proteins (IX, VII and X) involved in the extrinsic pathway of coagulation. Recombinant techniques combined with limited proteolysis will be employed to prepare factors IX and VII in which the Gla and Gla plus EGF-1 domains have been removed. Using the mutant and chimeric proteins, we will perform direct binding the kinetic studies on the phospholipid surface and in solution phase in order to further define the regions in factor IXa that are important for its interaction with factor VIIIa. We will also examine a hypothesis that factor VIIa binds to tissue factor (TF) with a significantly higher affinity than factor VII and that the EGF2 domain of factor VII is essential for its activation by factors Xa, IXa and VIIa. The planned studies will be carried out using both soluble and relipidated TF. In case of soluble TF, studies will be performed in the absence and presence of phospholipid, since binding of factors IX and VII to the phospholipid is expected to influence the reaction kinetics. Furthermore, we will examine the presence of tight and low affinity Ca2+ -binding sites in the Gla domains of factors IC, VII and X by studying the metal binding properties of these proteins in which the two high affinity Ca2+-binding sites in the nonGla region have been eliminated by site directed mutagenesis. In other experiments, by site-directed mutagenesis, we will change the contact residues (identified by molecular modeling in the first and the second domains of tissue factor pathway inhibitor (TFPI) and in the protease domains of factor VIIa and factor Xa in order to understand the structural basis for the first domain of TFPI to bind to factor VIIa and for the second domain of TFPI to bind to factor Xa. The data obtained from the proposed studies is expected to significantly enhance our understanding of the molecular mechanisms in involved in the assembly of intrinsic factor X- activating complex, and in the initiation and regulation of the extrinsic pathway of coagulation. These studies could also lead to the design of arthitorombotoc reagents and inhibit the TF-induced the TF-induced coagulation.